PROJECT SUMMARY/ABSTRACT Rett syndrome, an X-linked neurodevelopmental disorder caused predominantly by mutations in the gene encoding chromatin modulator Methyl-CpG-binding protein 2 (MECP2), is a leading genetic cause of disability in girls worldwide. Affected individuals develop typically for a period of 6-18 months, at which time the disease causes developmental regression with loss of purposeful hand movements, loss of speech, and autistic features. Treatment options are currently limited to symptomatic management, making the development of novel therapeutic approaches critically important. 10% of disease-causing MECP2 mutations arise in its C- terminal domain. However, despite the high prevalence of mutations in this region, few studies have investigated their pathophysiological mechanism(s). Furthermore, proposed therapeutic strategies for RTT focus largely on remedying loss-of-function (LOF) mutations that occur in other domains of MeCP2 - the methyl-binding and NCoR-interaction domains. Previous work suggests that the C-terminus may alter local chromatin binding and conformation, but thorough functional studies of different types of C-terminal mutations are lacking. Preliminary data shows that one of the most common C-terminal mutations that eliminates the entire domain, R294X, yields a truncated protein product in mice that accumulates with age and binds chromatin more tightly than wild-type MeCP2, suggesting that the R294X mutation may not act through simple LOF. With the resulting hypothesis that some C-terminal mutations cause Rett syndrome via a non-LOF mechanism, this proposal aims to 1) determine the contributions of the MeCP2 C-terminus to protein function as a transcriptional modulator, 2) elucidate how the R294X mutation elicits transcriptional dysregulation in three different brain regions, and 3) establish the behavioral consequences of transgenic complementation of the R294X allele. The proposed studies will involve the development of an in vitro framework to probe C- terminal Mecp2 mutations in functional assays for chromatin binding and co-repressor interaction, as well as the use of dual molecular and behavioral approaches to dissect the pathogenic mechanism of the prevalent R294X mutation. This research will provide mechanistic insight into the complex neurodevelopmental disorder Rett syndrome, as well as assist clinical decision-making by identifying the viability of proposed therapies for individuals with C-terminal MECP2 mutations. The proposed research will be accomplished through a carefully crafted fellowship training plan that involves opportunities to develop the applicant's technical expertise, critical-thinking skills, and scientific communication skills. Further, the research environment provided in turns by the MSTP, Vanderbilt Brain Institute, and Department of Pediatric Neurology is ideal for the proposed research and training, harboring a reputation for scientific excellence, a collegial community, and a strong focus on trainee development.